Assay system

ABSTRACT

The present invention relates to materials and methods for performing assays directed to monitoring activity and function of intracellular components, such as proteins associated with organelles and other intracellular structures. In particular, the invention relates to permeabilised cell preparations and their use in studying activity of intracellular components, in particular for studying sarcoplasmic reticulum function.

FIELD OF THE INVENTION

The present invention relates to materials and methods for performingassays directed to monitoring activity and function of intracellularcomponents, such as proteins associated with organelles and otherintracellular structures. In particular, the invention relates topermeabilised cell preparations which retain intracellular activity overlong periods of storage, and their use in studying activity ofintracellular components. In preferred embodiments the invention relatesto materials and methods for studying sarcoplasmic reticulum function,which are particularly useful in research directed to designing drugsfor heart disease and studying potential cardiac side-effects of drugsfor other conditions.

BACKGROUND OF THE INVENTION

Heart Failure is a common clinical problem with high morbidity andmortality. Left ventricular dysfunction (LVD) is present in around 3% ofall adults, and is symptomatic in around half of this group (McDonagh etal, 1997). Gwathmey et al (1987) were the first to suggest that abnormalCa²⁺ homeostasis in ventricular myocytes contributed to mechanicaldysfunction in heart failure.

Subsequently, many studies in both human and animal models of heartfailure have found reduced rate of decline of intracellular Ca²⁺transients e.g. (Bing et al, 1991; Beuckelmann et al, 1992; Anversa etal, 1991). Pieske et al (Pieske et al, 1995) found reduced peak systolicCa²⁺ levels which correlated with a negative force-frequencyrelationship in failing human trabeculae, and similar findings have beenreported in other human and animal studies (Beuckelmann et al, 1992; Qinat al, 1996).

One focus of study has been the function of the sarcoplasmic reticulum(SR) in heart failure. Schwinger et al (Schwinger at al, 1995) found 50%lower Ca²⁺ uptake rates in isolated SR vesicles and reducedSarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA2) activity in humandilated cardiomyopathy. Similar findings have been reported in otherhuman studies (Hasenfuss et al, 1994) and in animal models (e.g. (Afzal& Dhalla, 1992; Cory et al, 1993) using both tissue homogenates andvesicle preparations.

Protein abundance measurements for SR Ca²⁺ handling proteins areconflicting (see (Hasenfuss, 1998) and (Movsesian et al, 1989) forreview). The majority of abundance measurements indicate a reduction inSERCA2 protein expression in parallel with Ca²⁺ uptake (Mercadier et al,1990; Arai et al, 1993; Kiss et al, 1995; Matsui et al, 1995; Bartel etal, 1996; Currie & Smith, 1999). However, in human heart failure,reduced phosphorylation of phospholamban rather than reduced abundancemay be a factor (Movsesian et al., 1989).

With regard to the SR Ca²⁺ release channel/ryanodine receptor (RyR2),some studies have shown no difference in protein expression (Movsesianet al., 1994) while others indicate reduced expression in heart failure(Meyer et al., 1995; Brillantes et al., 1992).

Measurements of RyR2 function using laser-scanning confocal microscopyhave identified a reduced efficiency of coupling between Ca²⁺ influx andCa²⁺ release from the SR in a rat model of cardiac hypertrophy but noobvious change in RyR2 function (Gomez et al., 1997). Yet recent workhas shown that binding of the modulatory protein FKBP12.6 to RyR2 isreduced in human heart failure (Marx et al., 2000) and animal models(Ono et al., 2000). Reduced binding of FKBP12.6 to RyR2 affects thekinetics of the channel and therefore may alter excitation-contraction(E-C) coupling in failing hearts. However, total Ca²⁺ flux through theRyR2 in intact cardiac SR from failing hearts has not been examineddirectly. The remaining SR Ca²⁺ flux (non-RyR2 mediated Ca²⁺ leak) canpotentially affect SR function.

The pre-eminence of heart disease in the western world has meant thatthe majority of large pharmaceutical companies have active researchprograms to develop novel drugs for the cardiovascular system. Recentwork has highlighted a key defect in the failing heart as beingpredominately caused by the reduced effectiveness of the subcellularorganelle, SR, in the heart muscle cell. This organelle is of moregeneral interest to the pharmaceutical industry because the dysfunctionof the SR is a known trigger of potentially lethal arrhythmias (Bers2001). Recently legislation has been put in place that necessitates thescreening of all drugs for their arrhythmogenic potential. However,despite being the focus of research, there is a paucity of experimentalpreparations to allow the investigation of SR function.

SUMMARY OF THE INVENTION

The present inventors have appreciated that there is a need for an assaysystem that will facilitate research and drug design for heart disease.Given the strong evidence that sarcoplasmic reticulum (SR) function isan important factor when investigating heart failure, the inventors haveconcentrated on designing an assay system to follow SR activity, but thesystem is equally applicable to studying the activity and function ofother intracellular organelles such as the nucleus and mitochondria, andstructures such as the contractile proteins, and may also be applied tocell types other than myocytes. The system enables preparation of largebatches of material which can be stored for long periods of time withoutsignificant loss of activity and which lend themselves particularly wellto use in assays having a medium- or high-throughput format.

Existing assay systems for investigating SR function generally usemembrane vesicle preparations that are not in their naturalconfiguration and consequently have limited applicability to the intactcell. Typically a biochemical preparation of the SR is generated byhomogenisation and centrifugation to produce a vesiculated form of theSR. The capacity of the SR to sequester Ca²⁺ is measured using aradioactive Ca²⁺ tracer or the generation of a metabolite that was theresult of Ca²⁺ uptake (inorganic phosphate) (Coll et al 1999). A numberof disadvantages are associated with this sort of preparation including:(a) the process of isolating the SR vesicles is time consuming andexpensive; (b) using radioactive tracers is difficult and expensive; (c)the SR vesicle preparation does not remain viable, i.e. the activity ofthe preparation decreases progressively over the time course of theassay (approximately 30 minutes); and (d) the activity of thepreparation is not sustained well even when stored at −80° C.

In one aspect, the present invention provides methods of preparingfrozen stocks of cells, in particular permeabilised cells, in which theactivity of organelles and other intracellular structures can bemaintained during long periods of storage (up to 6 months, or even 9months or longer) and which lend themselves well to use in medium- andhigh-throughput assay systems.

In medium- or high-throughput assays, it may be desirable to automate asmany steps as possible while keeping the amount of manipulation requiredto a minimum. The cell preparations of the invention may be used easilyand routinely in assays of 96-well format or higher. Typically, amedium-throughput assay will involve tests of 100 to 200 differentcompounds in a test system, with a relatively detailed data acquisitionphase, e.g. establishing a time course for a particular effect. Ahigh-throughput assay will typically involve 10,000 or more compounds,but may involve less detailed data acquisition, e.g. looking for thepresence of absence of a desired effect.

Thus the invention provides a method for preparing a stock of cells forfuture use in an assay for determining activity or function of a targetintracellular component, the method comprising contacting a populationof cells with an effective amount of a permeabilising agent and freezingthe cells. The cells may either be incubated with the permeabilisingagent before freezing, such that they are permeabilised when frozen, orthey may be frozen immediately, such that permeabilisation continuesduring the freezing and/or thawing processes.

The permeabilised cells may be used in any assay which requires one ormore key-components of the assay medium to pass into the interior of thecell, e.g. to the cytosol or an intracellular organelle or structure.The key component may be required for a particular reaction to occur(e.g. it may be a substrate or co-factor for that reaction), or may berequired to measure the progress of a particular reaction (e.g. anindicator or reporter molecule of some kind).

Also possible, although less common, is that an assay may require a keycellular component to be able to pass into the assay medium, e.g. inorder to prevent its concentration increasing substantially within thecell as a result of a given reaction, possibly thereby inhibiting thatreaction. Thus, although the following will refer exclusively to a keycomponent of the assay medium, this term should be construed toencompass cellular components which are required to pass out of the cellinto the assay medium.

Typically, the component in question will be substantially unable tocross the cell's plasma membrane at the concentration used in the assay.Therefore the cells must be permeabilised so that the component to beable to pass through the membranes of enough of the cells in thepreparation for the assay to give meaningful results.

Use of permeabilised cells has the advantage of preserving theorganelles etc. in a physiological configuration. The minimal disruptionof the cell's structure and biochemistry means that the results from thefinal assay are more relevant to the intact cell than those from avesicular assay, and consequently can be relied on confidently.

The cells are typically mammalian cells, and are preferably myocytes(i.e. muscle cells), more preferably cardiac myocytes (including bothventricular and atrial myocytes) or skeletal myocytes. However thetechnique is suitable for use with any nucleated cell (e.g. anymammalian cells except red blood cells).

The permeabilising agent is preferably selective for the plasma membraneover the intracellular organelle membranes. This allows it topermeabilise the plasma membrane of the cells without significantlypermeabilising the membranes of the intracellular organelles such as thenucleus, endoplasmic or sarcoplasmic reticulum, or mitochondria.Preferably the organelle membranes are left intact and substantially notpermeabilised at all. Further, the plasma membrane should be leftsufficiently intact that the overall structural integrity of the cell ismaintained, e.g. organelles such as mitochondria are retained within thecell.

In order to achieve this selective permeabilisation, the permeabilisingagent may bind a membrane component of which the plasma membrane has ahigher content than the organelle membranes. For example, it may bind tocholesterol, as the plasma membrane has a higher cholesterol contentthan organelle membranes. Suitable permeabilising agents includesaponins, with beta-escin being particularly preferred. These moleculesprecipitate cholesterol, thus removing it from the membrane andincreasing its permeability. Other permeabilising agents may also beused, including various bacterial toxins, such as streptolysin-O andalpha-toxin from Staphylococcus aureus.

The present inventors have found that these permeabilising agents areeffective at permeabilising the cell's plasma membrane over a relativelynarrow range of concentrations. For example, in preferred embodimentsbeta-escin is added to a concentration of between 1 and 100 mg/ml, morepreferably between 1 and 50 mg/ml. At 1 mg/ml, only about 10% of asample of cardiac myocytes will be permeabilised, so more preferably aconcentration of between about 5 and 20 mg/ml will be used, morepreferably about 10 mg/ml.

Preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 990of the cells should be permeabilised. In preferred embodiments at least90% of the cells should be permeabilised, and preferably approximately100%.

Thus the required proportion of the cell population is sufficientlypermeable that the key component of the assay medium has access to thecytosol of those cells. It is generally sufficient to regard each cellas either permeable or not permeable to the key component(s) of theassay medium, and to determine the proportion of cells within thepopulation as a whole which can be said to be permeable.

The concentration of permeabilising agent required to achieve therequired degree of permeabilisation may vary depending on the commercialsource or batch of the permeabilising agent, or the type of cells to bepermeabilised.

The skilled person will be able to determine an appropriateconcentration of permeabilising agent to use in any particular case. Forexample, a population of the relevant cell type may be contacted withone or more concentrations of the permeabilising agent, preferably arange of concentrations. The cell population is also contacted with atest agent whose presence within the cell or transit across the membranecan be measured. The proportion of the cell population which displayspermeability of the plasma membrane to the test agent at eachconcentration of the permeabilising agent may then be determined. Theminimum concentration of the permeabilising agent which provides therequired degree of cell permeabilisation may then be used in thepreparation of the frozen cell stock.

The test agent may be any molecule which is substantially unable tocross an intact cell membrane (i.e. is substantially unable to cross themembrane in the absence of the permeabilising agent) and whose presencewithin the cell or transit across the cell membrane is detectable.Examples include dyes and other molecules whose presence within cells isdetectable visually, spectrophotometrically, or by other suitable means.Examples include trypan blue and fluorescent dyes. Calcium ions are notnormally able to cross the plasma membrane from the surrounding medium,but passage of calcium into the cell may be detected by a reportermolecule whose spectrophotometric properties (e.g. fluorescenceproperties) change on binding calcium, such as Fluo-3.

Alternatively, the test agent may cause a detectable change in cellmorphology or other physiologically detectable change in the cell. Forexample, calcium ions cause myocytes to contract from a rod-likemorphology to a more spherical morphology. This change may be used todetermine the proportion of permeabilised myocytes in a givenpopulation.

The Appendix below provides examples of the use of trypan blue andcalcium ions in assays to determine effective concentrations forparticular permeabilising agents in relation to rabbit cardiac myocytes.The skilled person will be able to adapt that teaching to any desiredcell type and assay system.

The permeabilising agent may be rendered substantially ineffective orinactivated by an appropriate dilution of the thawed cell preparationbefore performing an assay. A dilution of about ten-fold is preferred.This would be applicable, for example, to a preparation of cardiacmyocytes having a starting concentration of beta-escin of about 10mg/ml.

Thus, a dilution may be carried out directly on the thawed cellpreparation or as the preparation thaws. In other words, a dilution maybe carried out on the just thawed or thawing cell preparation withoutany procedures or manipulations being carried out on the preparationfirst, i.e. without any intervening procedures or manipulations.

For example, a diluent may be added directly to the just thawed cellpreparation, or directly to the partially frozen or completely frozencell preparation, which may assist thawing.

Accordingly, an assay may be performed on such a diluted cellpreparation, alternatively to being performed on an undiluted thawedcell preparation.

It will be seen that this greatly simplifies the preparation of theeventual assay, because no complicated procedures are necessary toremove or inactivate the permeabilising agent before beginning theassay.

Thus, it is not necessary for cells to be isolated from othercomponent(s) of the cell preparation before use in an assay. Forexample, it is not necessary for cells to be collected and washed in asuitable medium followed by resuspension in an assay medium for theassay. Therefore, an assay may be performed directly on a just thawedcell preparation or a diluted cell preparation without any procedures ormanipulations being carried out on the preparation first, i.e. withoutany intervening procedures or manipulations, as described in more detailbelow.

The cell density in the frozen preparation can be chosen according tothe dilution required to inactivate the permeabilising agent, as well asthe total number of cells and the cell density required for the assay.For example, an assay may require a density of approximately 10⁵, 10⁶ or10⁷ cells per ml. Thus the frozen cell preparation may have a celldensity of 10⁶, 10⁷ or 10⁸ cells per ml so that a 10-fold dilutionprovides the required density in the assay. For example, in the examplesdescribed below, the frozen cell preparation has a density of 1×10⁷cells per ml, which is diluted to 1×10⁶ cells per ml to perform SRcalcium flux assays.

The frozen cell preparation is preferably stored below −70° C., morepreferably below about −80° C. At temperatures of −80° C., sarcoplasmicreticulum activity in a preparation of permeabilised cardiac myocytescan be maintained for 6 months or more.

The pH of the cell preparation is typically buffered between about pH6.8 and about pH 7.4. This will normally be achieved by inclusion of anorganic buffer, such as Tris, or any of the “Goods buffers”, includingHEPES, BES, TES or PIPES.

The identity and concentration of the buffer may depend on the use towhich the cell preparation is to be put after thawing. In general,cellular metabolism has an acidifying effect on the solution. As aresult, it is desirable to use at least buffers at a concentration ofabout 5 mM or more, preferably about 25 mM, but up to about 50 mM ifrequired. The buffer may be present at up to 100 mM if it has an anionicresidue (e.g. HEPES), in which case the anionic buffer may be present asthe major anion in the solution.

It will be recognised that other components of the system may also havebuffering activity.

The cell preparation typically also comprises background electrolytes.Preferably the preparation comprises one or more monovalent metalcations, such as potassium and/or sodium ions, along with one or moresuitable, non-toxic anion. Preferably potassium ions are present atabout 100 mM, but may range in concentration from about 50 mM to about200 mM. Sodium ions, if present, are preferably at from about 5 mM toabout 40 mM.

Positively charged species are typically not present in total at morethan about 300 mM. Likewise, negatively charged species are typicallynot present in total at more than about 300 mM. Thus the concentrationsof electrolytes may be chosen depending on the concentations of othercharged species in the solution, such as the buffer (see above),chelating agents, etc.

Anionic electrolytes may include ions such as chloride, preferablypresent at about 30 mM to about 40 mM, as well as aspartate, glutamate,methyl-sulphate or any other suitable anion. The skilled person will becapable of selecting suitable electrolytes and determining suitableconcentrations thereof.

Other components may be present in the system depending on the intendeduse of the cell preparation when thawed. Many of these components couldbe added to the cell preparation after thawing, but may alternatively bepresent in the frozen cell preparation to minimise the number ofdifferent components to be added after thawing the cell preparation, andso reduce complexity of manipulation.

For example, it may be desirable that the cells contain substantially nofree calcium ions. This may be achieved by washing the cells afterpermeabilisation. Additionally or alternatively a calcium-specificchelating agent may be added to the solution before or after freezing. Asuitable agent is EGTA. Any suitable salt may be used, although apotassium salt is preferred.

A calcium-specific chelating agent may be particularly useful where thecells are to be used for an assay involving calcium flux, such as asarcoplasmic reticulum calcium flux assay. Typically exogenous calciumwill be added to the cells after thawing to perform such an assay.Preferably the concentration of chelating agent is such that it chelatesall the endogenous calcium ions present in the cells, but does notunduly affect the concentration of any such exogenously added calcium.For a cell preparation which is to be diluted 10-fold prior to theassay, a suitable concentration (e.g. of EGTA) is up to about 0.1 mM.Preferably the concentration is about 0.05 mM. Other calcium chelatingagents may be used; the concentration used may be adjusted in order togive similar chelation to any particular amount of EGTA.

Cell preparations to be used in assays which rely on an ATPase activitymay contain ATP. Examples of such assays include those relying oncalcium uptake by the SR, which is mediated by the sarco-endoplasmicreticulum ATPase (SERCA). Typically at least 1 mM ATP will be present ina cell preparation which is to be diluted 10-fold prior to commencementof the assay. Preferably about 5 mM ATP is present. However less, e.g.about 0.3 mM to 0.5 mM ATP may be present if the assay solution alsocontains creatine phosphate (see below).

Any salt of ATP may be used, although the sodium salt is preferred. Themagnesium salt is preferably not used, because Mg²⁺-ATP is the substratefor the SERCA. The operator can therefore control precisely when theassay is to begin by adding exogenous magnesium ions to the system. Thusif the frozen cell preparation is to be used for a calcium flux assay,the cell preparation is preferably kept substantially free of magnesiumions. This may be achieved by including EDTA or another Mg²⁺ chelatingagent in the cell preparation.

Creatine phosphate may be present in the cell preparation to regenerateATP from ADP. This may help to prevent ADP formed (by hydrolysis of ATP)during an assay from inhibiting an ATPase. Preferably between 3 mM and20 mM creatine phosphate is present in a solution to be diluted 10-foldbefore commencement of an assay. More preferably, a concentration ofbetween about 10 mM and about 15 mM is used.

In an assay for measuring calcium uptake by an organelle (e.g. the SR),it may be desirable to include an agent capable of precipitating calciumfrom solution at a suitable concentration of calcium. A suitableprecipitting agent will form a precipitate with calcium in the organelleand hence prevent the free calcium concentration in that organelle fromachieving levels at which influx of calcium is inhibited. Therefore theprecipitating agent should form precipitates at a calcium concentrationabove that of the assay medium, in order, not to interfere with thecalcium ions outside the relevant organelle. Oxalate is one example.Fluorides and phosphates should be avoided, because of the toxicity offluorides, and the further biological activities associated withphosphate ions.

A concentration of oxalate of approximately 20 mM in the final assaybuffer will maintain the calcium solution in the SR of a myocytepreparation at below about 10 μM. A concentration of between about 5 mMand about 30 mM oxalate may therefore be used in a cell preparation. Forcell preparations which are to be diluted prior to an assay it ispreferred not to concentrate the oxalate in the cell preparation, butinstead to use a diluent having the same concentration of oxalate.

Any suitable salt may be used, the potassium salt being preferred.

To minimise the effects of endogenous protein kinase activity on thetarget proteins (e.g. SERCA, Plb, RyR) of the assay, one or more proteinkinase inhibitors may be included in the cell preparation. For example,it may be desirable to include an inhibitor of cAMP-dependent proteinkinase. Additionally or alternatively, where the cell preparation is tobe diluted prior to an assay, the protein kinase inhibitor can be addedto the diluent. A preferred kinase inhibitor is H89 (source), which ispreferably used at 5-50 W, more preferably 10-30 μM, more preferablyapproximately 20 μM. H89(N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide, which isreadily commercially available) is particularly effective againstcAMP-dependant kinase in particular. However, in certain assays, it maybe desirable to preserve the kinase activity of the preparation, inwhich case kinase inhibitors can be omitted.

Typically substantially no cryoprotectant is present in the cellpreparation. These agents are conventionally used in thecryopreservation of proliferating cells to prevent the cells fromrupturing. However, such components are unnecessary in the preparationsof the present invention in which it is necessary for the cells to bepermeabilised, having been thawed. Commonly used cryoprotectants areglycerol and DMSO. Others include ethylene glycol, propylene glycol,hydroxy-ethyl-starch, polyvinylpyrrolidone and polyethlylene oxide.

Indeed, results described herein using cell preparations of theinvention show that activity of organelles and other intracellularstructures is preserved when freezing cell preparations in the absenceof conventional cryoprotectant, such as glycerol or DMSO (see, forexample, Table 1). This is surprising in view of the known difficultywith cryopreserving organelles in a fully functionally intact state.

Thus, in preparing a cell preparation of the invention there may besubstantially no cryoprotectant, for example, glycerol or DMSO, present,such that there may be substantially no cryoprotectant present in a cellpreparation of the invention.

For example, a cell preparation of the invention may contain cells, forexample mycocytes, such as cardiac myocytes; a permeabilising agent, forexample a saponin, such as beta-escin; a buffer, buffering thepreparation between about pH 6.8 and pH 7.4, for example an organicbuffer, such as HEPES at pH 7.0; background electrolytes, for example asource of monovalent metal cations, such as potassium ions, for exampleKCl; and substantially no cryoprotectant, such as glycerol or DMSO.

A cell preparation of the invention may contain cells, for examplemycocytes, such as cardiac myocytes; a permeabilising agent, for examplea saponin, such as beta-escin; a buffer, buffering the preparationbetween about pH 6.8 and pH 7.4, for example an organic buffer, such asHEPES at pH 7.0; background electrolytes, for example a source ofmonovalent metal cations, such as potassium ions, for example KCl; acalcium-specific chelating agent, for example EGTA; ATP; creatinephosphate; a low affinity Ca²⁺ precipitating agent, for example oxalate;and substantially no cryoprotectant, such as glycerol or DMSO.

Accordingly, as described above, it is not necessary for cells to beisolated from other component(s) of the cell preparation before use inan assay. For example, it is not necessary for cells to be collected andwashed in a suitable medium followed by resuspension in an assay mediumfor the assay. Therefore, an assay may be performed directly on a justthawed cell preparation or a diluted cell preparation without anyprocedures or manipulations being carried out on the preparation first,i.e. without any intervening procedures or manipulations as described inmore detail below.

This greatly simplifies the preparation of the eventual assay in whichthe cell preparation is used because no complicated procedures arenecessary to remove or inactivate a cryoprotectant before beginning theassay.

In a further aspect, the present invention provides a frozen cellpreparation as prepared by the methods described herein.

In a further aspect, the present invention provides a method ofdetermining an activity of a target intracellular component of apermeabilised cell, the method comprising providing a frozen cellpreparation of the invention, thawing said cell preparation, andperforming an assay for the activity of the target component.

In preferred embodiments, the invention provides a method of determiningan effect of a test substance on an activity of a target intracellularcomponent of a permeabilised cell, the method comprising providing afrozen cell preparation of the invention, thawing the cell preparation,contacting the thawed, permeabilised cells with said test substance, andperforming an assay for the activity of the intracellular structure ororganelle.

The target component may be a component (e.g. a protein) associated withan intracellular structure or organelle present in the relevant type ofpermeabilised cell. For example, a preparation of permeabilised myocytesmay be used to study myofibrils (contractile proteins), nuclei,mitochondria, or sarcoplasmic reticulum components.

In the instance of the SR, the change in activity is determined bydetecting changes in Ca²⁺ levels following contact between the heartcell and the substance under test. Within the sarcoplasmic reticulum,the protein in question may be the Ca²⁺ ATPase (SERCA), phospholamban(Plb), ryanodine receptor (RyR), FKBP12.6, Sorcin, Calmodulin,Ca-calmodulin-activated kinase, or cAMP-activated kinase.

The assay conditions can be easily adapted to study specific proteintargets. This is because regulatory proteins are still retained withinthe system, thus allowing the assay to include test agent interactionwith these auxiliary proteins.

The cell preparation may be contacted with a diluent prior to beginningthe assay, to form an assay mixture. The diluent may perform any or allof a number of roles. It may dilute the permeabilising agent in thepreviously-frozen cell preparation to a concentration at which it issubstantially ineffective. It may dilute the cells of the cellpreparation to the required cell density for the assay to be performed.It may adjust the concentration of other components in thepreviously-frozen cell preparation to the required value for the assay.It may provide components not present in the frozen cell preparationwhich are required to perform the assay. The frozen cell preparation maybe thawed prior to addition of the diluent, or the diluent may be addedto partially or completely frozen cells in order to assist the thawingprocess.

Thus, a diluent may be added directly to the thawed cell preparation oras the preparation thaws. In other words, a diluent may be added to thejust thawed or thawing cell preparation without any procedures ormanipulations being carried out on the preparation first, i.e. withoutany intervening procedures or manipulations. For example, a diluent maybe added directly to the just thawed cell preparation, or the partiallyfrozen or completely frozen cell preparation.

Thus, where a cell preparation contains ten times the concentration ofparticular components required for a given assay, the cell preparationmay be contacted with nine volumes of a diluent lacking thosecomponents.

Other components of the diluent (e.g. buffers, electrolytes, etc.) mayalso be present in the cell preparation. In preferred embodiments, anycomponents present in both cell preparation and diluent are equimolar inthe two solutions.

In preferred embodiments the diluent does not contain permeabilisingagent, and is used to dilute the permeabilising agent in the cellpreparation to a level at which it is substantially inactive orineffective.

Accordingly, an assay may be performed on such a diluted cellpreparation or assay mixture, alternatively to being performed on anundiluted thawed cell preparation.

In particular, an assay may be performed directly on any of these cellpreparations. In other words, an assay may be performed on any of thesecell preparations without any procedures or manipulations being carriedout on the preparation first, i.e. without any intervening procedures ormanipulations. For example, an assay may be performed directly on thediluted cell preparation, or the just thawed cell preparation.

It will be seen that this greatly simplifies the preparation of theassay, because no complicated procedures are necessary before beginningthe assay. Thus, it is not necessary for cells to be isolated from othercomponent(s) of the cell preparation before use in an assay. Forexample, it is not necessary for cells to be collected and washed in asuitable medium followed by resuspension in an assay medium for theassay. Therefore, an assay may be performed directly on a just thawedcell preparation or a diluted cell preparation without any procedures ormanipulations being carried out on the preparation first, i.e. withoutany intervening procedures or manipulations. As will be appreciated bythose skilled in the art, intervening procedures or manipulations whichare avoided relate to preparation of the cells to make them ready foruse in an assay, for example, to remove compounds the presence of whichmay be detrimental in an assay. Procedures or manipulations appropriateto the particular assay being carried out may be performed, for example,adding a test substance the effect of which is to be assessed in anassay as described above, or adding an initiating agent to start theassay as described below.

Thus, the method may comprise thawing the cell preparation andperforming an assay for the activity of the target component directly onthe thawed cell preparation, without first isolating the cells fromother component(s) of the cell preparation, for example by washing andresuspending the cells.

The method may comprise thawing the cell preparation, contacting thecell preparation with a test substance and performing an assay for theactivity of the target component, to determine the effect of the testsubstance on the activity of the target component, directly on the cellpreparation, without first isolating the cells from other component(s)of the cell preparation, for example by washing and resuspending thecells.

The method may further comprise contacting the cell preparation with adiluent to form an assay mixture and performing an assay for theactivity of the target component, which may be to determine the effectof a test substance on the activity of the target component, directly onthe assay mixture (diluted cell preparation), without first isolatingthe cells from other component(s) of the cell preparation, for exampleby washing and resuspending the cells.

The method may comprise adding one or more initiating agents in order tobegin the assay. The initiating agent may be a substance which isrequired for activity of the target component. The initiating agent may,for example, be a substrate for the target component, or for a componentupstream of the target component in a reaction pathway or cascade. Theinitiating agent may be added as part of the diluent, or separately,e.g. after the cell preparation and diluent have been incubated togetherfor a period of time to allow complete thawing of the cells.

For example, in an assay dependent on SERCA activity, the initiatingagent may be magnesium ions. As explained above, these can combine withATP already present in the assay mixture to form Mg²⁺-ATP, the substratefor the SERCA.

Other components may be added, in the diluent or separately, e.g.inhibitors of components whose activity would affect the assay. Forexample, an assay to monitor Ca²⁺ release from the SR may make use of aCa²⁺-ATPase inhibitor, such as thapsigargin. Inhibition of the ATPasewill prevent released calcium ions from being taken back up into the SR.Thus the rate of accumulation of calcium ions observed in the mediumwill provide a direct indication of the rate of release from the SR.Likewise, an assay for calcium uptake by the SR may utilise inhibitorsof calcium release from the SR, such as ruthenium red. Any calcium fluxassay will typically use an indicator for the presence of calcium ions,e.g. an agent capable of producing a detectable signal on contact withcalcium. Various suitable fluorescent dyes are known, including Fluo-3.Such components may also be present in the cell preparation, if desired.

Thus, in preferred embodiments, a complete assay system can be set up bysimple addition of an aliquot of diluent to an aliquot of frozen cells,optionally followed by a single addition of an initiating agent to theresulting assay mixture. This simplicity is particularly appropriate formedium/high-throughput assays, and/or automated assays.

In a further aspect, the present invention provides a kit comprising afrozen cell preparation as prepared by the methods described herein, incombination with a diluent as described in relation to the methodsabove. Optionally the kit comprises instructions for performing an assayaccording to any, appropriate aspect of the invention.

It will be apparent from the foregoing that it is also possible topermeabilise the cells after thawing. Thus, in a variation of earlieraspects of the invention, there is also provided a method fordetermining an activity of an intracellular structure or organelle of apermeabilised cell, the method comprising providing a frozen cellpreparation, thawing said cell preparation to produce a thawed cellpopulation, contacting the thawed cell population with a permeabilisingagent to produce a population of permeabilised cells, and performing anassay for the activity of the intracellular structure or organelle.

Also provided is a method for determining an effect of a test substanceon an activity of an intracellular structure or organelle of apermeabilised cell, the method comprising providing a frozen cellpreparation, thawing said cell preparation to produce a thawed cellpopulation, contacting the thawed cell population with a permeabilisingagent to produce a population of permeabilised cells, contacting saidpermeabilised cells with said test substance, and performing an assayfor the activity of the intracellular structure or organelle.

Preferred features of these methods are as described above in relationto earlier aspects of the invention. In particular, in these variationsan assay may be performed directly on the permeabilised cells, asdescribed above.

The following illustrates a use of the permeabilised cell preparationsof the invention. The present inventors have found that permeabilisedmyocytes can maintain Ca²⁺ uptake capacity for many hours and can beused in conjunction with a fluorescent Ca²⁺ indicator thereby allowingthe use of standard spectrometer equipment as a convenient andinexpensive way to measure Ca²⁺ uptake.

A preparation of frozen cardiac myocytes may therefore be providedaccording to the invention for analysis of sarcoplasmic reticulumcalcium flux.

Such a preparation may contain:

-   -   a) cardiac myocytes;    -   b) a source of monovalent metal cations, preferably potassium        ions, such as KCl or other background electrolyte;    -   c) ATP;    -   d) Creatine Phosphate;    -   e) organic buffer, e.g. at pH 7.0, such as HEPES;    -   f) EGTA;    -   e) low affinity Ca²⁺ precipitating agent, e.g. oxalate;    -   f) permeabilising agent, e.g. a saponin, such as beta-escin.

With this preparation of frozen cells may be provided one or both of thefollowing diluent solutions:

Diluent 1:

-   -   a) a source of monovalent metal cations, preferably potassium        ions, as found in the cell preparation;    -   b) ATP;    -   c) Creatine Phosphate;    -   d) organic buffer as found in the cell preparation;    -   e) EGTA;    -   f) low affinity Ca²⁺ precipitating agent as found in the cell        preparation, e.g. oxalate;    -   g) protein kinase inhibitor, e.g. H89.

This diluent is suitable for a SR calcium uptake assay as described inthe Examples. An assay mixture is prepared by adding 9 volumes ofdiluent to 1 volume of cell preparation. All components present in bothcell preparation and diluent are equimolar in the two solutions. Eachcomponent may have a concentration as set out above, or as in theExamples. Magnesium ions may be added to the assay medium to initiateSERCA activity. These may be present in the diluent, or may be providedseparately, in the kit if required, e.g. in the form of MgCl₂.

The diluent and/or the cell preparation may also comprise an agent whichprovides a detectable signal on contact with calcium ions, e.g. afluorescent dye such as Fluo-3.

It may also contain one or more agents for inhibiting release of calciumfrom the SR, such as ruthenium red.

The uptake assay may be used to determine substances under test which a)act directly or indirectly to alter the phosphorylation of phospholambanas this will enhance Ca²⁺ uptake; b) substances that interfere with theinteraction between phospholamban and the Ca²⁺ ATPase; c) substancesthat act directly on the Ca²⁺ ATPase to enhance or inhibit the activityof the enzyme; and d) substances that act on the Ca²⁺ leak from the SRvia the Ca²⁺ release channel (ryanodine receptor) or via independentleak pathways.

Diluent 2:

-   -   a) a source of monovalent metal cations, preferably potassium        ions, as found in the cell preparation;    -   b) ATP;    -   c) Creatine Phosphate;    -   d) organic buffer as found in the cell preparation;    -   e) EGTA;    -   f) low affinity Ca²⁺ precipitating agent as found in the cell        preparation, e.g. oxalate;    -   g) MgCl₂;    -   h) SERCA inhibitor, e.g. thapsigargin;    -   i) protein kinase inhibitor, e.g. H89.

This diluent is suitable for a SR calcium release assay as described inthe Examples. As with diluent 1, all components present in both cellpreparation and diluent are equimolar in the two solutions, and may haveconcentrations as set out above, or as in the Examples. An assay mixtureis prepared by adding 9 volumes of diluent to 1 volume of cellpreparation.

The diluent and/or the cell preparation may also comprise an agent whichprovides a detectable signal on contact with calcium ions, e.g. afluorescent dye such as Fluo-3.

This assay may be used to determine substances under test which a) actdirectly or indirectly to alter Ca²⁺ extrusion via the ryanodinereceptor; b) substances that interfere with the interaction between theryanodine receptor and the various modulatory proteins e.g. FKBP12.6,sorcin, c) substances that may alter Ca²⁺ fluxes by changes in thephosphorylation status of the ryanodine receptor; and d) substances thatact directly on Ca²⁺ leak pathways that are independent of the ryanodinereceptor.

For both the uptake and release assays, the amount of Ca²⁺ in saidsolution can be measured either by following a time course of the Ca²⁺levels, e.g. by measuring every 1 to 2 seconds for up to 10 minutes; orby measuring the level of Ca²⁺ at a set time.

These assays can be applied to a multi-well format (e.g. 96 well, 384well, or more) which is suitable for standard fluorescence platereaders.

In this form, the SR preparation can be used to profile a very largenumber of candidate compounds. For example, in the context of an uptakeassay, a high through-put screen can be used for >10,000 test compounds,to provide a read-out that shows whether a compound stimulates orinhibits SERCA mediated uptake. A medium through-put screen would allow100-200 compounds to be screened quickly while examining the time courseof uptake. This type of read-out would give a more detailed read-out ofthe test compound's action, i.e. whether the compound increased maximumturn-over rate or the Ca²⁺ sensitivity of Ca²⁺ SERCA-mediated Ca²⁺uptake.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Ca²⁺ ATPase mediated uptake in permeabilised cell preparations.

A. Shows a sample trace of Ca²⁺ concentration within a suspension of5×10⁵ cells/ml (Fura-2 fluoresence) (SERCA activity assay). As indicatedabove the figure, Ca²⁺ was added to increase the [Ca²⁺] within theaggregate to ˜1.5 μM. No Ca²⁺ uptake occurs due to the absence of Mg²⁺(and therefore MgATP). Addition of Me starts the reaction. The initialincrease of [Ca²⁺] is caused by displacement of Ca²⁺ from ATP. The SRsubsequently takes up the Ca²⁺ within the cuvette, a process that isenhanced by addition of 10 uM cAMP.

B. Shows detailed SERCA parameters derived from complete time coursesignals.

C. Shows inhibition of SERCA by thapsigargin.

FIG. 2: Caffeine stimulated Ca²⁺ leak from permeabilised cell aggregates

Shows the principle of measuring the Ca²⁺ sensitive leak from cellaggregate preparations. Ca²⁺ uptake is stimulated by addition of analiquot of Ca²⁺. SERCA inhibition is induced by thapsigargin (5 uM).Ca²⁺ leak is stimulated by the addition of 40 mM caffeine and blocked byaddition of ruthenium red (as indicated).

FIG. 3: Concentration-response curve showing permeabilising effect ofCalbiochem saponin (0.01-1000 μg/ml) in 5 mM EGTA and 5 mM Ca²⁺ EGTA.

Permeabilisation was quantified using the Ca²⁺-contraction assay andcounts were normalised against the control count. Each point is themean±s.e. mean of 3-8 observations.

FIG. 4: Concentration-response curve showing cell counts in 5 mM EGTAand 5 mM Ca²⁺ EGTA exposed to Sigma saponin at 1-1000 μg/ml. Resultswere assessed using the Ca²⁺-contraction assay and normalised againstthe control count. Each point is the mean±s.e. mean of 6 observations.

FIG. 5: Concentration-response curve showing the permeabilising effectof β-escin (0.01-10000 μg/ml) in 5 mM EGTA and 5 mM Ca²⁺ EGTA. Resultswere quantified using the Ca²⁺-contraction assay and normalised againstthe control count. Each point is the mean±s.e. mean of 3-10observations.

FIG. 6: Concentration-response curve showing the permeabilising effectof Calbiochem saponin (0.1-10000 μg/ml) in 5 mM EGTA. Results werequantified using the access of trypan blue and normalised against thecontrol count. Each point is the mean±s.e. mean of 3 observations.

FIG. 7: Concentration-response curve showing the permeabilising effectof β-escin (0.1-10000 μg/ml) in 5 mM EGTA. Results were quantified usingthe access of trypan blue and normalised against the control count. Eachpoint is the mean±s.e. mean of 3 observations.

FIG. 8: Concentration-response curves for (a) Calbiochem saponin(0.01-10000 μg/ml), (b) Sigma saponin (1-1000 μg/ml) and (c) β-escin(0.01-10000 μg/ml) in 5 mM EGTA and 5 mM Ca²⁺ EGTA. Results wereassessed using the Ca²⁺-contraction assay and normalised against theinitial count. Each point is the mean±s.e. mean of 3-10 observations.

FIG. 9: Fit of the non-linear logistic curves for each agent andassociated assay method.

ED₅₀ values were estimated from this fit and are shown along side eachplot. Each point is the mean±s.e. mean of 3-10 observations.

FIG. 10: Ca²⁺ ATPase mediated uptake in permeabilised cellpreparations—Example data from assays with test compounds.

Characteristics of Ca²⁺ uptake were analysed in the presence of varioustest compounds in assays using a multi-well format. Example dataachieved with inhibitory and stimulatory compounds are shown.

Table 1: Sustained activity from cell aggregates.

Myocyte aggregates were held in storage for approximately 6 months ormore at −80 C using the procedure described below in the Examples. Ondefrosting the characteristics of Ca²⁺ uptake were analysed in terms ofthe maximum rate of uptake (Vmax) and the sensitivity of Ca²⁺ uptake(Km). Neither parameter showed significant deterioration over thestorage period. Values are expressed as mean+/−SEM.

Table 2: Stock solutions.

This Table shows the volume of each stock solution required to producethe range of concentrations of permeabilising agent in 1 ml of cellsuspension.

Table 3: ED₅₀ values.

ED₅₀ values for each agent studied for Ca²⁺-contraction and trypan blueassay methods.

Table 4: Ca²⁺ ATPase mediated uptake in permeabilised cellpreparations—pharmacological characterisation.

Frozen cell preparations were used to analyse characteristics of Ca²⁺uptake in the presence of various stimulators or inhibitors of SERCA.Results for sensitivity of Ca²⁺ uptake (Km) are shown. Values areexpressed as mean+/−SEM.

EXAMPLES Materials and Methods Cell Isolation

The procedure is based on the principle that cells within the intactheart can be isolated by gradual removal from their associatedconnective tissue and cellular syncitium. Before isolation, thoroughcleaning of perfusion equipment was carried out. 2-3 1 of doubledistilled water was perfused through the system followed by 1-2 1 ofsterile water. New Zealand White rabbits (2-2.5 kg) were given anintravenous injection of 500 U heparin together with an overdose ofsodium pentobarbitone (100 mg/kg). The hearts were rapidly excised,weighed and cannulated onto a Langendorff perfusion column via theaorta. The hearts were perfused in a retrograde fashion at a rate of 25ml.min⁻¹ (37° C.), initially with 150 ml of Krebs Henseleit solutionwith the following composition (mM): 120 NaCl, 20 HEPES, 5.4 KCl, 0.52NaH₂, PO₄, 3.5 MgCl₂.6H₂0, 20 Taurine, 10 Creatine, 11.1 Glucose; pH 7.4with NaOH at 37° C. [Ca²⁺] within this solution is in the order of 6-7μM due to Ca²⁺ contamination. This solution aids in washing away bloodand reduces the probability of clot formation. Taurine may aid incardio-protection against Ca²⁺ paradox (Chapman & Tunstall, 1983).Thereafter, the hearts were perfused for 1 min with Krebs Henseleitsolution supplemented with 1.4 mg ml⁻¹ collagenase (type 1, WorthingtonChemical Co.), 0.1 mg ml⁻¹ protease (type XIV, Sigma Chemical Co.) and afurther addition of 50 μM CaCl₂ to activate the enzymes. After a time,such that the enzyme solution had fully perfused the heart and equipmentthe enzyme containing solution was re-circulated for a further 6 min.The heart was then perfused with a 0.1% Bovine Serum Albumin (BSA) KrebsHenseleit solution with the addition of 62 μM CaCl₂. The BSA containingsolution provides extra substrate for superfluous enzyme. The atria wereremoved and discarded whereas the right and left ventricle was kept forexperiments.

Intact Cell Preparation

The chosen ventricle was carefully cut into small pieces and placed intoa culture flask containing 20 ml of the 0.1% BSA containing KrebsHenseleit solution described above. This solution also contained 0.125mM CaCl₂. The suspension was gently shaken for 30-60 min. After theappropriate time the solution containing the cardiomyocytes was removed.The suspension was subjected to gentle centrifugation and the pelletre-suspended into 0.1% BSA Krebs Henseleit solution containing 0.125 mMCaCl₂. Cells were then washed in the following solution: (mM) 100 KCl, 5Na₂ATP, 10 Na₂Creatine Phosphate, 5.5 MgCl₂, 25 HEPES, 1 K₂EGTA, pH 7.0(20-21° C.)

Preparation of Cells for Freezing

Intact cells within the above solution were counted using ahaemocytometer and adjusted to a concentration of 1×10⁶/100 ul (i.e.1×10⁷ cells.ml⁻¹) in intracellular solutions (S1 or S2 depending on thepurpose of the assay: S1: (mM) 100 KCl, 5 Na₂ATP, 10 Na₂CreatinePhosphate, 25 HEPES, 0.5 K₂EGTA, pH 7.0 (20-21° C.), 20 oxalate. S2:(mM) 100 KCl, 5 Na₂ATP, 5.5 MgCl₂, 10 Na₂Creatine Phosphate, 25 HEPES,0.5 K₂EGTA, pH 7.0 (20-21° C.), 20 oxalate.

Finally the 0.1 mg.ml⁻¹ beta-escin (Sigma) was added and the preparationwas immediately aliquoted into cryovials (at 1×10⁶ cells) and placedinto a ‘Mr Frosty’ container overnight to allow gradual freezing. Thesewere then placed into liquid nitrogen for freezing.

Unfreezing of Cell Preparation

900 μl of S1 or S2 diluion solution as appropriate was added to the 100μl of cryovial cell preparation.

Composition of S1 Dilution Solution

S1: (mM) 100 KCl, 5 Na₂ATP, 10 Na₂Creatine Phosphate, 25 HEPES, 0.05K₂EGTA, 0.01 K₄Fluo-3, pH 7.0 (20-21° C.), 20 K₄Oxalate, 0.005 RutheniumRed, 0.05 mM H89.

Composition of S2 Dilution Solution

S2: (mM) 100 KCl, 5 Na₂ATP, 5.5 MgCl₂, 10 Na₂Creatine Phosphate, HEPES,0.05 K₂EGTA, 0.01 K₄Fluo-3, 0.005 Thapsigargin, pH 7.0 (20-21° C.), 20K₄Oxalate, 0.05 mM H89.

Results SR Uptake Assay

(e.g. an assay for SR Ca²⁺ ATPase activity and modulation byphospholamban)

Aliquots of cells are defrosted gradually with the addition of 9 volumesof S1 solution. This was followed by addition of CaCl₂ to increase thefree [Ca²⁺] to ˜1.5 μM (˜100 μM total Ca²⁺). The mixture can then bedistributed into the 96 or high format arrays. Incubation of thepreparation with the compound of interest can occur for 15-20 mins andthe SR uptake reaction began by addition of 5.5 mM MgCl₂ (withstirring). This provides the substrate (MgATP) to allow SR uptake ofCa²⁺. There are two options for measurement: (i) follow the time courseof the Ca²⁺ uptake profile in detail by measuring every 1-2 s for up to10 mins (ii) measuring the Ca²⁺ concentration at a set time (e.g. 10mins) after initiation of the reaction. Compounds that stimulate the SRCa²⁺ ATPase will cause the decline of Ca²⁺ concentration to occur fasterthan control (vehicle). Conversely compounds that inhibit the SR Ca²⁺ATPase will slow the decline. These effects can be monitored in detail(option (i)—see FIG. 1 and description thereof for an example) or bysimply measuring Fluo-3 fluorescence at one time point (option (ii)).

This Assay is Sensitive to:

1. Compounds acting directly or indirectly to alter the phosphorylationof phospholamban, which will enhance Ca²⁺ uptake.2. Compounds that interfere with the interaction between phospholambanand the Ca²⁺-ATPase.3. Compounds that act directly on the Ca²⁺ ATPase to enhance or inhibitthe activity of the enzyme.4. Compounds that act on the Ca²⁺ leak from the SR via the Ca²⁺ releasechannel (ryanodine receptor) or via independent leak pathways.

Note that screening for compounds that interfere with ryanodine receptorleak can be differentiated by using Ruthenium Red (1-5 μM); thiscompound blocks Ca²⁺ release via the ryanodine receptor. This lattereffect can also be detected by a second type of assay configuration (seebelow).

SR Ca²⁺ Release Assay

(e.g. to assess the rate of Ca²⁺ loss from the SR both via the ryanodinereceptor and by independent routes.)

This assay requires the preparation stored in solution S2 (see above).The cell aggregate preparation is defrosted in 9× volumes of S2 with theaddition of 2-3 aliquots of 100 μM CaCl₂. The preparation is thenaliquoted into the high format wells (96 or 384 well). The reaction isinitiated by addition of a single aliquot of the SR Ca²⁺ ATPaseinhibitor thapsigargin (or any other established selective Ca²⁺ ATPaseinhibitor). The slow progressive increase in Ca²⁺ concentration withinthe bath reflects Ca²⁺ loss from the SR both via the ryanodine receptorand via independent leak pathways. Ryanodine receptor mediated leak canbe enhanced for purposes of drug screening by inclusion of caffeine(5-10 mM) to the reaction mixture. As with the assay system above, twooptions for measurements are possible (i) follow the timecourse of theCa²⁺ release profile in detail by measuring every 1-2 s for 10 mins (ii)measuring the Ca²⁺ concentration at a set time (e.g. 10 mins) afterinitiation of the reaction. Compounds that stimulate the SR Ca²⁺ leakwill cause the increase of Ca²⁺ concentration to occur faster thancontrol (vehicle). Conversely compounds that inhibit the SR Ca²⁺ leakwill slow the increase of Ca²⁺ concentration. These effects can bemonitored in detail (option (i)—see FIG. 2 and description thereof foran example) or by simply measuring Fluo-3 fluorescence at one time point(option (ii)).

This Assay is Sensitive to:

-   -   1. Compounds acting directly or indirectly to alter Ca²⁺        extrusion via ryanodine receptor.    -   2. Compounds that interfere with the interaction between the        ryanodine receptor and the various modulatory proteins e.g.        FKBP12.6, sorcin.    -   3. Compounds that may alter Ca²⁺ extrusion by changes in the        phosphorylation status of the ryanodine receptor.    -   4. Compounds that act directly on Ca²⁺ leak pathways that are        independent of the ryanodine receptor.

Note that screening for compounds that interfere with ryanodine receptorleak can be differentiated by using Ruthenium Red (5-10 μM) thiscompound blocks Ca²⁺ release via the ryanodine receptor.

Permeabilised myocytes prepared as described above retain significantactivity even after 6 months or more at −80° C. On defrosting thecharacteristics of Ca²⁺ uptake were analysed in terms of the maximumrate of uptake (Vmax) and the sensitivity of Ca²⁺ uptake (Km). Neitherparameter showed significant deterioration over the storage period.Values are shown in Table 1, expressed as mean+/−SEM.

APPENDIX Determination of Concentrations for Permeabilising Agents

Commercial preparations of permeabilising agents such as saponins(including beta-escin) may vary significantly in activity betweensources and batches. For this reason, it may be desirable to test eachnew batch of permeabilising agent in order to determine a suitableconcentration for any given assay.

The following description illustrates two methods which were used totest permeabilisation of rabbit myocytes by a batch of beta-escin, andto compare two different commercial batches of saponin.

Methods Preparation of EGTA and Ca²⁺ EGTA Solutions

Permeabilisation of myocytes occurred in mock intracellular bathingsolutions. This ensured that there were no major changes inintracellular ions and metabolites once the sarcolemma was madehyperpermeable.

Two HEPES-buffered Krebs-based bathing solutions were produced; one withhigh calcium (Ca²⁺ EGTA), the other with low calcium (EGTA). The twosolutions had the same basic composition (mM): KCl 100 (BDH LaboratorySupplies, Poole, England; lot no: A154338 907), NaCl 10 (Sigma-AldrichChemicals, Germany; lot no: 01500), MgCl 5.5 (BDH Laboratory Supplies,Poole, England; lot no: 39627), HEPES 25 (Sigma Chemical Company, St.Louis, USA; lot no: 90K5406), ATP 5 (disodium salt; Sigma Chemical Co.,St. Louis, USA; lot no: 100K7051), creatine phosphate 10 (disodium salttetrahydrate; Fluka Chemicals; lot no: 399292/1 40201) and either EGTAor Ca²⁺ EGTA both 5 (Sigma Chemical Co., St. Louis, USA: lot no:119H5433).

The volume was made up to 100 mls using distilled water and pH wasadjusted to exactly 7.0. 20 mls of each solution was pipetted intoindividual labelled plastic containers (to prevent reaction of ATP withglass). These were frozen (approximately −80° C.) and stored untilrequired. The calcium concentrations of 5 mM EGTA and 5 mM Ca²⁺ EGTAsolutions were calculated at <1 nM and 20 μM respectively.

Cell Counting

The haemocytometer method of cell counting was used to count the numberof non-permeable myocytes before and after application of thepermeabilising agent (either saponin or β-Escin). Freshly dissociatedcardiac muscle cells were obtained in a solution of low calcium. Thesewere filtered and transferred into two 13.5 ml test tubes. Each wascentrifuged for approximately 12 seconds at 5 g and the supernatant wasdiscarded. Approximately 4.5 ml of EGTA solution was used to re-suspendthe pellet in each tube. The contents of both were combined to form thestock suspension.

An initial count was performed on the stock suspension to ensure thatthe cell count was sufficiently high. In our experience 25×10⁴ cells perml of suspension was found to be the lowest initial count which wouldyield distinguishable results after permeabilisation, so theconcentration of the stock suspension was altered until it lay abovethis.

All cell counting described in this appendix was performed using ahaemocytometer.

Ca²⁺-Contraction Assay

Physiologically, myofilaments are activated by a rapid increase in thecytosolic Ca²⁺ concentration from 0.1 μM to approximately 2 μM whichoccurs mainly due to Ca²⁺ release from the SR (Levick, 2000). TheCa²⁺-contraction assay used in this investigation relies upon the factthat cells with permeabilised membranes allow free access of Ca²⁺ to thecytosol when exposed to high extracellular Ca²⁺ concentrations. Thisactivates the myofilaments of the cell causing it to contract into aball. Non-permeabilised myocytes retain their characteristic rod shape.The effects of permeabilising agents on the cell population werequantified by counting the proportion of rod-shaped cells.

Trypan Blue Assay

Access of the vital dye Trypan blue (MW: 950 approx.) into myocytes wasused to evaluate permeabilisation by Saponin (Calbiochem brand) orβ-escin. Permeabilised myocytes were identified by positive staining(blue colour) of the cell contents. Cells with intact membranes excludedthe dye and it was this population that was counted.

Preparation of Permeabilising Agent Solutions

On each experimental run only one permeabilising agent was investigated.Since both saponin and β-escin degrade in solution within approximatelythree to four hours it was necessary to make up fresh solutions for eachrun. Saponin (Calbiochem®, USA; lot no:B31340) or β-Escin (SigmaChemical Co., St. Louis, USA; lot no:109H0964) were made-up in threestock concentrations, 100 mg/ml, 10 mg/ml and 10 μg/ml (0.01 mg/ml) indistilled water. The quantities of each stock added to obtain therequired concentrations when added to 1 ml of cell suspension are shownin Table 2.

Experimental Protocol Ca²⁺-Contraction Assay

On each run utilising the Ca²⁺-contraction assay, sixteen 13.5 ml testtubes were obtained. Eight of these were labelled “EGTA” whilst theremaining eight were labelled “CaEGTA”. Within both sets of eight,individual tubes were labelled control 1, control 2, 0.01, 0.1, 1, 10,100 and 1000.

The cell stock suspension was agitated to ensure uniform distribution ofcells and exactly 1 ml was pipetted into each tube labelled “EGTA”.These tubes were then transferred to the fridge to prevent degradationof the cells.

Each tube was put through the following protocol in turn. The requiredvolume of permeabilising agent was added (see Table 2) and a stopclockwas started. During the two minute incubation time, exactly 0.5 mls ofthe contents of the “EGTA” tube was withdrawn using a pipette andtransferred to the corresponding “CaEGTA” tube.

Once the incubation time had elapsed the tubes were centrifuged forapproximately 12 seconds at 5 g and the supernatant was withdrawn. The“EGTA” pellet was re-suspended in 0.5 mls of EGTA solution and the“CaEGTA” pellet in 0.5 mls of CeEGTA solution. Both tubes were agitatedand the cell density determined by haemocytometer. The protocol wasfollowed for all concentrations under investigation; for the twocontrols no permeabilising agent was added and thus no incubation stepwas required.

Trypan Blue Assay

For each run utilising the trypan blue assay, eight 13.5 ml test tubeswere obtained. Each was labelled control 1, control 2, 0.1, 1, 10, 100,1000 or 10000.

The cell stock suspension was agitated to ensure uniform distribution ofcells and exactly 1 ml was pipetted into each tube. These were thentransferred to the fridge to prevent degradation of the cells. Each tubewas put through the following protocol in turn. The required volume ofpermeabilising agent was added (see Table 2) and a stopclock wasstarted.

Once the incubation time had elapsed the tube was centrifuged forapproximately 12 seconds at 5 g and the supernatant was withdrawn. Thepellet was re-suspended in 0.5 mls of EGTA solution and 0.5 mls of 0.5%Trypan blue (BDH Laboratory Supplies, Poole, England) in 5 mM EGTA,which had been previously gently heated to ensure that the dye wasdissolved. The tube was agitated and cell density determined byhamocytometer counting.

The protocol was followed for all concentrations under investigation;for the two controls no permeabilising agent was added and thus noincubation step was required.

Exposure to High Concentrations

The effect of exposing myocytes to exceedingly high concentrations ofpermeabilising agent (10000 μg/10 mg) was investigated for both β-Escinand saponin. The protocol followed was the same as the Ca²⁺-contractionassay protocol (described above). 100 μl of the 100 mg/ml stock wasadded to the 1 ml suspension volume to achieve the requiredconcentration (see Table 2). A control plus a low concentration of agent(1 μg/ml) were also studied to validate results.

Comparison of Two Brands of Saponin

To demonstrate the variation in effect of saponins from differentmanufacturers, two saponins were investigated; one from Calbiochem(Calbiochem®, USA; lot no:B31340), as used previously, and one fromSigma (Sigma Chemical Co., St. Louis, USA; lot no:19H7841). Theconcentrations investigated were 1, 10, 100 and 1000 μg/ml for eachsaponin plus two controls. The experimental protocol followed was thesame as for the Ca²⁺-contraction assay method above.

Confocal Imaging of Cell Permeabilisation

In order to demonstrate the process of permeabilisation, images weretaken using laser-scanning confocal microscopy and fluo-3 (MolecularProbes). Fluo-3 is a calcium sensitive fluorescent indicator; local Ca²⁺concentration is directly proportional to fluorescence intensity. Thetime-course of permeabilisation was measured by the entry of fluo-3 intothe cell. Excitation of fluo-3 occurred at 488 nm whilst emission wasmeasured at 518 nm. Cells were viewed with a 60× water objective lensand Sigma saponin added to give a final concentration of 100 μg/ml.Images were taken every two seconds. The time from addition ofpermeabilising agent to fully permeabilised state was noted.

Statistical Analysis of Results

All experimental results were expressed as the mean±s.e. mean of nexperiments. These were normalised against either (i) control counts or(ii) initial counts.

Graphs were plotted and non-linear logistic curves were fitted usingleast squares technique by the Microcal™ Origin™ package (version 5.0,Microcal Software Inc.). ED₅₀ values and their associated errors werealso estimated using this software. Analysis of variance (ANOVA) usingTukey's post-test comparison was used to verify significance betweenED₅₀ values (GraphPad InStat™, GraphPad Software, V2.05a).

Results Ca²⁺-Contraction Assay

A decrease in rod count in 5 mM Ca²⁺ EGTA indicated that the sarcolemmawas hyperpermeable to the high calcium concentration of the bathingmedium; this is due to activation of the myofilaments by the highintracellular Ca²⁺ concentration and the development of ahyper-contraction.

In general, exposure of isolated myocytes to the saponins or β-escincaused the rod count to decrease in a concentration-dependent manner.

Calbiochem Saponin

The results show that Calbiochem saponin produced permeabilisation overthe concentration range 10-10000 μg/ml for the Ca²⁺-contraction assaymethod (FIG. 3). This was evidenced by the large difference between therod counts in 5 mM EGTA and 5 mM Ca²⁺ EGTA. Between the concentrations0.01-10 μg/ml, mean counts in 5 mM EGTA and 5 mM Ca²⁺ EGTA were similar;error bars overlapped and therefore means were not consideredsignificantly different (n=4-8). A significant decrease in the rod countin 5 mM Ca²⁺ EGTA was apparent at 10 μg/ml and, over a ten-fold increasein concentration from 10-100 μg/ml, the rod count dropped byapproximately 80%. The calculated ED₅₀ value for Calbiochem saponinusing this assay system was 45.4±16.2 μg/ml. Approximately 100% of theinitial cell population were hyperpermeable at 1000 (n=8) and 10000μg/ml (n=3). The minimum effective concentration for Calbiochem saponinwas 100 μg/ml. A slight trend to increase was noted in the counts in 5mM EGTA with increasing saponin concentrations.

Sigma Saponin

The permeabilising effects of Sigma saponin were studied over theconcentration range 1-1000 μg/ml; results are presented in FIG. 4. Fromexperience, this was the range over which the permeabilising effect ofsaponin occurred. Sigma saponin began to hyper-contract, hencepermeabilise, the myocytes between 1-10 μg/ml (ED₅₀=5.9±0.4 μg/ml).Approximately 100% of the cells were permeabilised at 100 and 1000μg/ml. The minimum effective concentration of Sigma saponin was 100μg/ml. Counts in 5 mM EGTA over the range were stable at approximately100% (1.0) thus consistent with the control count. Comparison with theCalbiochem brand showed a significant difference (p<0.05).

β-escin

Rod counts were stable in both 5 mM EGTA and 5 mM Ca²⁺ EGTA over theconcentration range 0.01-1 μg/ml (n=7-10); these are presented in FIG.5. The first concentration of β-escin which began to causepermeabilisation was 10 μg/ml (n=6), this was the steepest section ofthe graph and error bars were greatest here. Permeabilisation was seenbetween 1-10000 μg/ml; ED₅₀=8.3±1.1 μg/ml. When compared to the ED₅₀ forSigma saponin no significant difference occurred (p>0.05); in contrast asignificant difference was shown when compared to Calbiochem saponin(p<0.05). Approximately 100% of the control population werehyperpermeable to Ca²⁺ at 100, 1000 and 10000 μg/ml β-escin (n=3-7). Theminimum effective concentration of β-escin was 100 μg/ml.

Rod counts in 5 mM EGTA were maintained at the control level up to 100μg/ml (n=7); thereafter they declined over two concentration incrementsto zero; a Ca²⁺-independent contraction had occurred. The ED₅₀ value forthis effect was 1192.1±112.3 μg/ml.

Trypan Blue Assay

The access of trypan blue into the cell was assessed by plotting themean percentage of rods in 5 mM EGTA excluding dye at each concentrationstudied (0.1-10000 μg/ml; n=3). Rods with intact membranes excluded thedye and thus a reduction in the count reflected an increased proportionof rods with hyperpermeable membranes. In general, the number of cellsallowing trypan blue access increased as concentration ofsaponin/β-escin increased.

Calbiochem Saponin

Between 0.1-10 μg/ml the mean count was stable at approximately 90%(0.9) (FIG. 6). Thereafter the mean count began to decrease. At 1000 and10000 μg/ml approximately 100% of the control population were permeableto trypan blue. The ED₅₀ for this process was calculated as 46.4±0.9μg/ml and was not significantly different to the ED₅₀ value forCalbiochem saponin using the Ca²⁺-contraction assay.

β-escin

At 0.1 μg/ml mean rod count was approximately 90% (0.9); this increasedat 1 μg/ml to approximately 110% (1.1) (FIG. 7). However, error bars forthese two points overlap and so means are not considered to bedifferent. Trypan blue began to enter the myocytes from 1-10 μg/ml; at10 μg/ml the mean rod count had decreased by approximately 35% of thecontrol value. Approximately 100% of the control population werepermeable to dye at 100, 1000 and 10000 μg/ml thus the minimum effectiveconcentration of β-escin was 100 μg/ml. The ED₅₀ for access of trypanblue was calculated as 13.9±2.2 μg/ml β-escin; this was notsignificantly different from the ED₅₀ value for β-escin using theCa²⁺-contraction assay.

Reduction in Cell Numbers Independent of Permeabilising Agent

The cell quality and integrity of their membranes was thought to be amajor variable in the study. In addition it was recognised that theprotocol itself may have disrupted some of the membranes. To account forthese factors the mean count for each concentration was normalisedagainst the initial count. The range 0.01-1 μg/ml was chosen todetermine this effect as no decrease due to permeabilisation wasdemonstrated over this range in the results normalised against thecontrol count. The results for Ca²⁺-contraction assay for Calbiochemsaponin, Sigma saponin and β-escin are shown in FIG. 8. The graphs showthat for the three agents studied an approximate 10-20% reduction incell count occurred in 5 mM EGTA whilst this increased to approximately50-65% in 5 mM Ca²⁺ EGTA.

Assessment of ED₅₀ Values

ED₅₀ values were estimated from the non-linear logistic curves (FIG. 9)using the standard logistic equation equation. The small Chi-squarevalues obtained (0.00003-0.012) indicate that the fit was reasonable inall cases.

The ED₅₀ is the concentration of agent which permeabilises 50% of thecontrol population. The ED₅₀ values for each agent and associated assaymethod are shown in Table 3.

REFERENCES

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1. A method for preparing a stock of cells for future use in an assayfor determining activity or function of a target intracellularcomponent, the method comprising contacting a population of cells withan effective amount of a permeabilising agent and freezing the cells. 2.A method according to claim 1 wherein there is substantially nocryoprotectant present in the cell preparation.
 3. A method according toclaim 2 wherein cryoprotectant is glycerol or DMSO.
 4. A methodaccording to claim 1 wherein the cells are myocytes.
 5. A methodaccording to claim 4 wherein the cells are cardiac myocytes.
 6. A methodaccording to claim 1 wherein the permeabilising agent is selective forthe plasma membrane over the intracellular organelle membranes.
 7. Amethod according to claim 6 wherein the permeabilising agent is asaponin.
 8. A method according to claim 7 wherein the saponin isbeta-escin.
 9. A method according to claim 1 wherein the permeabilisingagent may be rendered substantially ineffective by a ten-fold dilutionof the thawed cell preparation before performing the assay.
 10. A methodaccording to claim 1 comprising storing the cells at about −70° C. orbelow.
 11. A method according to claim 1 wherein the cell stock furthercomprises one or more of: i) an organic buffer; ii) a source ofmonovalent metal cations, preferably potassium ions.
 12. A methodaccording to claim 1 wherein the cell stock further comprises one ormore of: i) an organic buffer; ii) a source of monovalent metal cations,preferably potassium ions; iii) a calcium-specific chelating agent; iv)ATP; v) creatine phosphate; vi) a calcium precipitating agent; vii) aprotein kinase inhibitor.
 13. A frozen cell preparation, preparable bythe method of claim
 1. 14. A method for determining an activity of atarget intracellular component of a permeabilised cell, the methodcomprising providing a frozen cell preparation as prepared by the methodof claim 1, thawing said cell preparation, and performing an assay forthe activity of the target component.
 15. A method according to claim 14wherein the assay comprises the step of contacting the thawed,permeabilised cells with a test substance, and determining the effect ofsaid substance on the activity of the target component.
 16. A methodaccording to claim 14 wherein the assay for the activity of the targetcomponent is performed directly on the cell preparation.
 17. A methodaccording to claim 14 wherein the target component is associated with anintracellular structure or organelle.
 18. A method according to claim 17wherein the target component is associated with myofibrils, nuclei,mitochondria, or sarcoplasmic reticulum.
 19. A method according to claim14 wherein the change in activity is determined by detecting changes inCa²⁺ levels following contact between the permeabilised cells and thesubstance under test.
 20. A method according to claim 19 wherein thetarget component is the Ca²⁺ ATPase (SERCA), phospholamban (PIb),ryanodine receptor (RyR), FKBP12.6, Sorcin, Calmodulin,Ca-calmodulin-activated kinase, or cAMP-activated kinase.
 21. A methodaccording to claim 14 further comprising the step of contacting the cellpreparation with a diluent to form an assay mixture.
 22. A methodaccording to claim 21 wherein the method comprises addition of ninevolumes of diluent per volume of cell preparation.
 23. A methodaccording to claim 21 wherein the diluent does not containpermeabilising agent.
 24. A method according to claim 21 wherein theassay for the activity of the target component is performed directly onthe assay mixture.
 25. A method according to claim. 21 furthercomprising the step of adding an initiating agent to the assay mixtureto begin the assay, wherein the initiating agent is a substance which isrequired for activity of the target component.
 26. A method according toclaim 25 wherein the assay is dependent on SERCA activity and theinitiating agent comprises magnesium ions.
 27. A method according toclaim 26 wherein the assay monitors Ca²⁺ release from the SR and thediluent comprises a Ca²⁺-ATPase inhibitor.
 28. A method according toclaim 21 wherein the assay monitors calcium uptake by the SR and thediluent comprises an inhibitor of calcium release from the SR.
 29. Amethod according to claim 21 wherein the assay mixture comprises anindicator for the presence of calcium ions.
 30. A method according toclaim 29 wherein the indicator is a fluorescent. dye whose fluorescencechanges on contact with calcium.
 31. A kit comprising a frozen cellpreparation prepared by a method according to claim 1 and a diluent. 32.A kit according to claim 31 wherein the frozen cell preparationcomprises: g) cardiac myocytes; h) a source of monovalent metal cations,preferably potassium ions; i) ATP; j) Creatine Phosphate; k) organicbuffer, e.g. HEPES; 1) EGTA; g) low affinity Ca²⁺ precipitating agent,e.g. oxalate; h) permeabilising agent, e.g. a saponin.
 33. A kitaccording to claim 32 wherein the diluent comprises: a) a source ofmonovalent metal cations, preferably potassium ions, as found in thecell preparation; b) ATP; c) Creatine Phosphate; d) organic buffer asfound in the cell preparation; e) EGTA; f) low affinity Ca²⁺precipitating agent as found in the cell preparation, e.g. oxalate; g)protein kinase inhibitor, e.g. H89.
 34. A kit according to claim 32further comprising a source of magnesium ions.
 35. A kit according toclaim 32 further comprising an agent for inhibiting release of calciumfrom the SR.
 36. A kit according to claim 31 wherein the diluentcomprises: a) a source of monovalent metal cations, preferably potassiumions, as found in the cell preparation; b) ATP; c) Creatine Phosphate;d) organic buffer as found in the cell preparation; e) EGTA; f) lowaffinity Ca²⁺ precipitating agent as found in the cell preparation, e.g.oxalate; g) MgCl₂; h) SERCA inhibitor, e.g. thapsigargin; i) proteinkinase inhibitor, e.g. H89.
 37. A kit according to claim 31 furthercomprising an agent which provides a detectable signal on contact withcalcium ions.